Identical phosphatase mechanisms achieved through distinct modes of binding phosphoprotein substrate

Abstract

Two-component signal transduction systems are widespread in prokaryotes and control numerous cellular processes. Extensive investigation of sensor kinase and response regulator proteins from many two-component systems has established conserved sequence, structural, and mechanistic features within each family. In contrast, the phosphatases which catalyze hydrolysis of the response regulator phosphoryl group to terminate signal transduction are poorly understood. Here we present structural and functional characterization of a representative of the CheC/CheX/FliY phosphatase family. The X-ray crystal structure of Borrelia burgdorferi CheX complexed with its CheY3 substrate and the phosphoryl analogue reveals a binding orientation between a response regulator and an auxiliary protein different from that shared by every previously characterized example. The surface of CheY3 containing the phosphoryl group interacts directly with a long helix of CheX which bears the conserved (E - X(2) - N) motif. Conserved CheX residues Glu96 and Asn99, separated by a single helical turn, insert into the CheY3 active site. Structural and functional data indicate that CheX Asn99 and CheY3 Thr81 orient a water molecule for hydrolytic attack. The catalytic residues of the CheX.CheY3 complex are virtually superimposable on those of the Escherichia coli CheZ phosphatase complexed with CheY, even though the active site helices of CheX and CheZ are oriented nearly perpendicular to one other. Thus, evolution has found two structural solutions to achieve the same catalytic mechanism through different helical spacing and side chain lengths of the conserved acid/amide residues in CheX and CheZ.

Fig. 1.

Overall topology of . (A) Ribbon representation of the asymmetric unit. CheX is cyan and CheY3 is green. CheY3 Asp79 (orange), (purple), CheX Glu96 (red, Upper), and CheX Asn99 (red, Lower) are in stick representation. Mg²⁺ is a magenta sphere. (B).Overlay of B. burgdorferi CheX (cyan) and T. maritima CheX chain B (pdb 1XKO, tan). Regions of B. burgdorferi CheX with high disorder (Cα thermal B-factors > 70) are blue. B. burgdorferi CheX residues 37–40 are sketched as blue dots. T. maritima CheX residues corresponding to the disordered regions of B. burgdorferi CheX are red.

Fig. 2.

Comparison of the relative orientation of CheX and CheY3 with that of YPD1 (a histidyl phosphotransferase) and SLN1-R1 (a receiver domain) within (pdb 2R25). For (Upper), CheX is gray except for α1^′, which is cyan. CheY3 is green except α1 is blue and α5 is red. For (Lower), YPD1 is gray except for αC (teal). SLN1-R1 is pale yellow except α1 is blue and α5 is red.

Fig. 3.

Active site structure and kinetic characterization of CheX and CheY3. (A) Close-up view of the active site. Residues are green for CheY3 and cyan for CheX. The ordered water molecule (Wat B162) is a blue sphere and Mg²⁺ is a light green sphere. Hydrogen bonds revealed by the structure are represented by black-dashed lines. The red-dashed line between Wat B162 and the beryllium atom (distance of 3.4 Å) is nearly collinear with the bond connecting the beryllium atom and Asp79 OD1. (B) CheX phosphatase activities. The CheY3 concentration is 15 μM. Reactions contained wild-type CheX/wild-type CheY3 (closed squares), CheX 96EA/wild-type CheY3 (closed triangles), CheX 99NA/wild-type CheY3 (closed diamonds), or wild-type CheX/ CheY3 81TA (closed circles). Rates measured in the absence of CheY3 for wild-type CheX (open squares), CheX 96EA (open triangles) and CheX 99NA (open diamonds) reflects nonspecific phosphatase activity due to trace contaminants, and was subtracted out. (C) Schematic model for transition state stabilization in CheX -catalyzed dephosphorylation of CheY3. The bipyramidal transition state is colored black with dashed lines representing partial bonds. Stabilizing interactions are represented with red-dashed lines with the assumption that interactions observed in persist in the transition state. One of several possible hydrogen bonding arrangements between CheY3 T81 or CheX N99 and the water is shown. Residues from CheY3 (green) and CheX (cyan) are indicated.

Fig. 4.

Two structural solutions achieve the same catalytic end. (A) Ribbon representation of B. burgdorferi and E. coli (pdb 1KMI) with superposition of CheY3 (green) and CheY (light green). CheX is cyan and CheZ is orange. CheX Glu96 (Left) and Asn99 (Right) are in cyan sticks. CheZ Asp143 (Left) and Gln147 (Right) are in orange sticks. The moiety is in purple sticks. (B) Close-up of region within the indicated square from Panel (A). Select active site groups from B. burgdorferi CheY3 (green) or E. coli CheY (light green) are shown. The residues are sticks and the divalent cations (Mg²⁺ for CheY3 and Mn²⁺ for CheY) are spheres.